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Understanding Germination and Pathogenicity in Zygomycota Species Through Genomic and Transcriptomic Approaches by Poppy Sephton Clark

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Understanding Germination and Pathogenicity in Zygomycota Species through Genomic and Transcriptomic Approaches by Poppy Sephton Clark A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences University of Birmingham August 2019 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Mucorales spores are the causative agents of the emerging disease mucormycosis. Mucorales species are also responsible for high quantities of food spoilage annually. The mechanism by which Mucorales spores cause disease and rot relies upon spore germination, however the mechanism underlying germination in these species remains poorly understood. Presented here are results which characterise Mucorales spore germination, through phenotypic and transcriptional studies (RNA-Seq), which followed the defined germination phenotype throughout. Hallmark pathways are identified through analysis of differentially expressed genes and co-transcriptional networks, providing targets for germination inhibition. With the resulting transcriptional data, the genome of Rhizopus delemar was enriched and analysed, thus providing better information on the Mucoralean genome. Comparative genomics was also employed to better understand genotypic variation between Mucorales species. To examine the differences in pathogenicity between species, and assess the impact of germination stage on pathogenicity, the transcriptional profile (RNA-Seq) of selected Mucorales species was examined upon phagocytosis by innate immune cells. To better understand the corresponding host response, the transcriptional response (single cell RNA-Seq) of innate immune cells to Mucorales infection was also examined. Finally, germination targets identified through the described analyses were targeted with suspected inhibitors to confirm function in germination regulation. This work has furthered our basic understanding of germination in these ancient fungi, indicated pathways essential to the germination programme of Mucorales species, and demonstrated a crucial role played by many of these pathways in host-fungal interactions of the Mucorales. 1 Acknowledgements I would like to express my gratitude to Dr Elizabeth Ballou and Dr Kerstin Voelz, both advisors have offered incredible support, encouragement and guidance. A special thanks to Dr Voelz for taking me on and supporting me through the initial stages of my study, and to Dr Ballou for encouraging me to continue along my research path and offering amazing support and mentorship along the way. I would like to express my appreciation to Dr Christina Cuomo, with whom I spent invaluable time training with, and Professor Robin May, whose encouragement during my undergraduate studies led me to continue my research in the fungal field. And of course, a huge thanks to the amazing HAPI Lab members (past and present!). It has been a joy to work in such a positive environment, with enthusiastic scientists so supportive of one another! I would like to say a special thank you to Daniel, for all of his support, curiosity and encouragement. For celebrating triumphs with me, sharing frustrations with me and always encouraging me to go after my goals. It is truly appreciated! Last but not least, I would like to say thank you to my parents, for encouraging me for as long as I can remember! For encouraging curiosity and always showing an interest in whatever I was most fascinated with, thank you! Finally, I would like to say thank you to my Gran, for all of her encouragement along the way! Table of Contents Chapter 1: Introduction .................................................................................. 5 Introduction: Mucorales .................................................................................................... 5 Food Spoilage ............................................................................................................................ 8 Mucormycosis ............................................................................................................................ 9 Mucorales spores and germination regulation ......................................................................... 10 Germination as a mechanism of pathogenicity ......................................................................... 12 Project Aims ................................................................................................. 15 Literature Review ............................................................................................................ 16 Introduction to fungal morphotypes: Spores and Hyphae ........................................................ 16 Importance of spores and hyphae in pathogenicity and food spoilage ..................................................... 20 Spore Composition .................................................................................................................. 22 The spore cell wall ...................................................................................................................................... 22 Spore compartmentalization and dormancy factors .................................................................................. 24 Water availability and metabolic activity ................................................................................................... 25 The Spore Germination Program .............................................................................................. 26 Spore polarization ...................................................................................................................................... 26 Hyphal outgrowth and extension ............................................................................................................... 28 Regulation of Germination ....................................................................................................... 29 The nutritional environment and germination .......................................................................................... 30 Germination Regulation via Ph, Temperature, Light and Environmental Gases ......................................... 35 Signalling molecules ................................................................................................................................... 40 Materials and Methods…………..………………………………………………………………………………………..43 Microbial Culture ............................................................................................................. 44 Fungal Culture ......................................................................................................................... 44 Spore Isolation ............................................................................................................................................ 44 Spore Growth ............................................................................................................................................. 44 Endosymbiont Curing ................................................................................................................................. 44 Bacterial Culture ...................................................................................................................... 45 Germination Phenotyping ................................................................................................ 46 Spore Germination Assay ......................................................................................................... 46 Live cell Imaging ......................................................................................................................................... 46 Flow Cytometry .......................................................................................................................................... 46 XTT Assay .................................................................................................................................................... 46 Exosome Release ........................................................................................................................................ 47 Genomic DNA Extraction, Sequencing and Analysis ......................................................... 48 Genomic DNA Extraction .......................................................................................................... 48 Fungal DNA Extraction ................................................................................................................................ 48 Bacterial DNA Extraction ............................................................................................................................ 48 Genomic DNA Sequencing ........................................................................................................ 48 Fungal Sequencing .....................................................................................................................................
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